A number of extracellular stimuli, including soluble cytokines and insoluble matrix factors, are known to influence murine embryonic stem cell self-renewal and differentiation behavioral responses via intracellular signaling pathways, but their net effects in combination are difficult to understand. of Oct-4 expression levels. This data-driven, multivariate (16 conditions 31 components 3 time points Mouse monoclonal to beta Tubulin.Microtubules are constituent parts of the mitotic apparatus, cilia, flagella, and elements of the cytoskeleton. They consist principally of 2 soluble proteins, alpha and beta tubulin, each of about 55,000 kDa. Antibodies against beta Tubulin are useful as loading controls for Western Blotting. However it should be noted that levels ofbeta Tubulin may not be stable in certain cells. For example, expression ofbeta Tubulin in adipose tissue is very low and thereforebeta Tubulin should not be used as loading control for these tissues = 1,500 values) proteomic approach identified a set of signaling network components most critically associated (positively or negatively) with differentiation (Stat3, Raf1, MEK, and ERK), proliferation of undifferentiated cells (MEK and ERK), and proliferation of differentiated cells (PKB, Stat3, Src, buy 56-53-1 and PKC). These predictions were found to be consistent with previous literature, along with direct test here by a peptide inhibitor of PKC. Our results demonstrate buy 56-53-1 how a computational systems biology approach can elucidate key sets of intracellular signaling protein activities that combine to govern cell phenotypic responses to extracellular cues. Mouse embryonic stem (ES) cells are derived from the inner cell mass of preimplantation blastocysts (1). They are pluripotent and can contribute to every tissue in the adult organism (2). This developmental potential can be maintained in the presence of the gp130 signaling ligand, leukemia-inhibitory factor (LIF) (3, 4). Binding of LIF to the LIFR-gp130 receptor complex stimulates signaling through two main pathways, the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway and the mitogen-activated protein kinase kinase (MEK)/extracellular-regulated kinase (ERK) pathway (5). The functions of these LIF-stimulated pathways in maintenance of ES cell pluripotency are not completely comprehended, although activation of Stat3 buy 56-53-1 is usually indispensable for self-renewal (6, 7). Despite the importance of LIF in murine ES cell self renewal, maintenance of somatic stem cells has remained elusive, and ES cells from other species, buy 56-53-1 e.g., human, apparently cannot be maintained by the presence of LIF alone (8, 9). Moreover, extracellular matrix factors, such as fibronectin (Fn) and laminin (Ln), can significantly influence the effects of cytokines (10C12). Cytokines and matrix factors that regulate ES cell processes operate through multiple intracellular signaling pathways, which are generally found across diverse cell types (13). Indeed, cytokines that exert opposite effects on differentiation outcomes can activate comparable pathways (14). Conversely, more than one factor may be capable of triggering comparable cell responses, as is the case with LIF and IL-6 (15, 16), but the potency of any given factor may depend around the signaling context. As with other cell behavioral responses to cytokine and matrix factors, such as migration and apoptosis, the control of stem cell self-renewal versus differentiation responses most likely depends on the balance among a set of intracellular signaling activities, rather than being uniquely determined by one particular component. Although two new ES cell-specific regulatory transcription factors have been recently identified, Ehox and Nanog (17C19), it remains important to address the question of how the expression of transcription factors is regulated by extracellular cues through intracellular signals. An urgent question is: how can a critical set of intracellular signaling network activities be identified for governing stem cell self-renewal versus differentiation responses across a broad spectrum of extracellular cytokine and matrix cues? For instance, we study here Oct-4 expression levels in mouse ES cells after treatment with 16 different conditions, for three time points over 5 days, comprising permutations of cytokine [LIF and fibroblast growth factor 4 (FGF-4)] and matrix factor (Fn and Ln) combinations. Because so few signaling pathways have been explored in relation to this important problem, especially across cytokine/matrix combinations, a data-driven approach, analogous to the now-common transcriptional profiling methods for studies directed to genomic issues, is an attractive initial step. Because we are concerned with protein signaling network activities governing cell behavior, protein-level measurements involving multiple pathways must be made and analyzed. Therefore, we use quantitative Western blotting to obtain levels of 31 phospho-proteins involved in more than a half-dozen signaling pathways. Finally, many computational modeling techniques often used for genomic investigations, such as clustering, are not appropriate for.